Permeant Cations and Blockers Modulate pH Gating of ROMK Channels

Abstract

External potassium (K) activates the inward rectifier ROMK (K(ir)1.1) by altering the pH gating of the channel. The present study examines this link between external K and internal pH sensitivity using both the two-electrode voltage clamp and the perfused, cut-open Xenopus oocyte preparation. Elevating extracellular K from 1 mM to 10 mM to 100 mM activated ROMK channels by shifting their apparent pK(a) from 7.2 +/- 0.1 (n = 6) in 1 mM K, to 6.9 +/- 0.02 (n = 5) in 10 mM K, and to 6.6 +/- 0.03 (n = 5) in 100 mM K. At any given internal pH, the number of active ROMK channels is a saturating function of external [K]. Extracellular Cs (which blocks almost all inward K current) also stimulated outward ROMK conductance (at constant 1 mM external K) by shifting the apparent pK(a) of ROMK from 7.2 +/- 0.1 (n = 6) in 1 mM K to 6.8 +/- 0.01 (n = 4) in 1 mM K + 104 mM Cs. Surprisingly, the binding and washout of the specific blocker, Tertiapin-Q, also activated ROMK in 1 mM K and caused a comparable shift in apparent pK(a). These results are interpreted in terms of both a three-state kinetic model and a two-gate structural model that is based on results with KcsA in which the selectivity filter can assume either a high or low K conformation. In this context, external K, Cs, and Tertiapin-Q activate ROMK by destabilizing the low-K (collapsed) configuration of the selectivity filter.

Tertiapin-Q Blocks Recombinant and Native Large Conductance K+ Channels in a Use-Dependent Manner

“…Therefore, the blocking actions of tertiapin-Q are not specific to recombinant GIRK1ϩ GIRK4 heteromultimeric and ROMK1 and ROMK2 homomultimeric channels as previously proposed Lu, 1998, 1999;Kitamura et al, 2000;Sackin et al, 2003). Although the inhibition of GIRK and BK channels occur within a similar nanomolar range, the mechanisms of tertiapin-Q inhibition appear to be different among K ϩ channels.…”

“…Although the inhibition of GIRK and BK channels occur within a similar nanomolar range, the mechanisms of tertiapin-Q inhibition appear to be different among K ϩ channels. Inhibition of GIRK channels by tertiapin occurs within a minute and does not exhibit rapid recovery (Kitamura et al, 2000), whereas tertiapin-Q block of ROMK channels can take up to 10 min, and it is reversible with an overshoot of the outward conductance during washout (Sackin et al, 2003). In contrast, inhibition of BK channels is use-and voltage-dependent, requiring Ͼ15-min stimulation by depolarizing steps, and the recovery is slow.…”

“…Thus, there is substantial interest in identifying specific modulators of these ion channels. Tertiapin, a 21-amino acid-containing peptide isolated from the venom of the European honey bee (Gauldie et al, 1976;Xu and Nelson, 1993), has been characterized as a specific and potent blocker of recombinant GIRK1ϩGIRK4 heteromultimers and ROMK1 at nanomolar affinity (Jin and Lu, 1998) and ROMK2 at micromolar range (Sackin et al, 2003). On the other hand, tertiapin has been reported to be less effective on some Kir channels; for example, inhibition of Kir2.1 channels by 1 M tertiapin is less than 10% (Jin and Lu, 1998).…”

Inhibition of ROMK channels by low extracellular K+ and oxidative stress

“…In this way external K ϩ could antagonize a variety of inhibitory effects that act on the inside of the cell including cytoplasmic acidification, PIP2 depletion, and oxidative stress. Consistent with this idea, several Kir channels including Kir1.1 (ROMK) and 4.1 are selectively activated by cations that interact with the pore either as permeant ions or blockers (7,9,29). In this respect, regulation of Kir channels by external K ϩ is analogous to C-type inactivation of voltage-gated K ϩ channels, a process that is also inhibited by extracellular permeant ions (18,21).…”

“…] o downregulate the channels (7,28,29,31). One aspect of this response is a shift in the dependence of channel activity on intracellular pH, with low [K ϩ ] o moving the titration curve for inhibition of the channels toward a higher, more physiological pH range.…”